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Aug 23, 2013 - Here, we demonstrate the role of specific glycan–receptor interactions in the regenerative process using Hydra magnipapillata as a mo...
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Unique, Polyfucosylated Glycan−Receptor Interactions Are Essential for Regeneration of Hydra magnipapillata Sonu Sahadevan,† Aristotelis Antonopoulos,‡ Stuart M. Haslam,‡ Anne Dell,‡ Subramanian Ramaswamy,†,§ and Ponnusamy Babu*,§ †

Institute for Stem Cell Biology and Regenerative Medicine, NCBS-TIFR, Bangalore, 560065 India Department of Life Sciences, Imperial College London, London SW7 2AZ, U.K. § Glycomics and Glycoproteomics, Centre for Cellular and Molecular Platforms, NCBS-TIFR, Bangalore, 560065 India ‡

S Supporting Information *

ABSTRACT: Cell−cell communications, cell−matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan−receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N- and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan−receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan−receptor interactions in the regeneration of H. magnipapillata.

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Within each subclass of glycans, there is a huge heterogeneity, which makes structural characterization of these molecules complicated and requires multiple strategies. Nevertheless, recent technological advances in mass spectrometry and sample preparation have allowed a precise structural characterization of glycans from various sources such as glycoproteins, cells, tissues, organs, and whole animals. This is particularly beneficial in the case of structurally unusual and new glycans with a limited tissue source. Here, we use Hydra magnipapillata as a model organism to study the role of glycans in stem cell function and regenerative processes. The freshwater polyp Hydra, of the phylum Cnidaria, has a remarkable capacity to regenerate and possibly lacks senescence.14 Fully regenerated Hydra polyps can be formed from either dissociated cell aggregates or amputated body parts. Hydra, which has radial symmetry, consists of two germ layers, ectoderm and endoderm, with an intermediate extracellular matrix (ECM) called the mesoglea. Hydra possesses three stem cell lineages: ectodermal, endodermal, and interstitial. While the epithelial stem cell populations differentiate into head- and

ll eukaryotic cell surfaces are covered with glycans attached to proteins and lipids. A wealth of information is available on the biological roles of glycans in cell growth and development,1 immune recognition and response, cell−cell communication,2 tumor growth and metastasis,3 microbial pathogenesis,2 and cell migration. In addition to these functions, glycoconjugates such as glycoproteins, glycolipids, and glycosaminoglycans have been used as cell surface markers and implicated in proliferation, differentiation, self-renewal, and death of stem cells.4−6 For example, it has been established that polysialic acid-NCAM regulates the myelination, axon guidance, synapse formation, and functional plasticity of the nervous system.7 Chondroitin sulfate proteoglycans have been implicated in axon guidance, neurite outgrowth, and selfrenewal of neural stem cells.8 The interaction between selectins and sialyl LewisX has been shown to play important roles in homing and mobilization of hematopoietic progenitor cells.9 Furthermore, O-fucosylated glycans have been shown to be important for the regulation of Notch signaling during development,10 inhibition of neuronal and oligodendroglial differentiation, maintaining the neural stem cell pool, and promoting astroglial differentiation.11 The structure of glycans attached to glycoconjugates is complex and can vary from branched oligosaccharides (N- and O-glycans) to highly acidic, linear glycosaminoglycans.12,13 © 2013 American Chemical Society

Received: July 2, 2013 Accepted: August 23, 2013 Published: August 23, 2013 147

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Figure 1. MALDI-TOF MS analysis of H. magnipapillata reveals polyfucosylated N-glycans. N-Linked glycans from H. magnipapillata were isolated, permethylated, and analyzed by MALDI-TOF MS. Data represent partial MS spectra from the 50% MeCN fraction depicting only the most abundant family (for the less abundant family, see Figure 1 of the Supporting Information). All molecular ions are [M + Na]+. Putative structures are based on composition, tandem MS, and biosynthetic knowledge. Structures that show sugars outside of a bracket have not been unequivocally defined.

complex-type glycans (85−90%) with a minor abundance of high-mannose glycan structures (10−15%). With regard to the complex glycans, compositional analysis suggested the presence of multiple HexNAc residues bearing various amounts of fucose. When the N-glycans described above were subjected to MALDI-TOF/TOF MS/MS, analysis indicated the presence of two different families (Figures 2 and 3 of the Supporting Information). The first and most abundant family comprised multiantennary (bi- to penta-antennary) N-glycans; their mannose branches were extended with HexNAc−HexNAc moieties (Figure 1). Within this family, the most abundant Nglycans corresponded to bi-, tri-, tetra-, and penta-antennary structures having attached principally two fucose residues on each HexNAc residue [i.e., m/z 3544, 4731, 5918, and 7104, respectively (Figure 1 and Figure 2 of the Supporting Information)]. Lower in abundance, but within the same family, were structures having more than four fucoses per HexNAc−HexNAc antenna reaching even up to six fucose residues, with more fucoses being on the terminal nonreducing HexNAc residue (up to four fucose residues; i.e., m/z 4241, 5775, and 7310). However, none of the antennae of the Nglycan family described above was found to be extended with poly-HexNAc−HexNAc repeating units. The second, lowerabundance, family detected (Figures 1 and 3 of the Supporting Information) was characterized by the presence of an additional HexNAc residue on the nonreducing side of the HexNAc− HexNAc antenna, with the latter being substantially less fucosylated bearing up to only one fucose per HexNAc residue. Attempts to elucidate the sequence and linkage of the polyfucosylated epitopes using commercially available α-L-

foot-specific tissues, the multipotent interstitial stem cells give rise to nerve cells, germ cells, nematocytes (stinging cells), and gland cells.15 In addition to various cell types, the outer surface of the Hydra ectoderm is covered with a layer of glycocalyx, whose structure and composition have recently been investigated.16 Cell−cell communication, cell migration, and cell−mesoglea interactions have been shown to be important for Hydra regeneration and reaggregation.17−19 However, the molecular players involved in these events have not been fully characterized. In particular, the structures of glycans attached to proteins and their interacting partners are completely unknown. Here, an ultrasensitive, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry-based analysis of N- and O-glycans revealed that the Hydra glycome predominantly consists of novel, polyfucosylated LacdiNAc structures. A complete lack of regeneration of Hydra body parts is observed when the polyfucose glycan− receptor interaction is disrupted as shown by complementary experiments with fuco-lectins and a global metabolic inhibitor of fucosylation. Identification of a putative polyfucose binding endogenous C-type lectin supports the hypothesis that glycan− receptor interaction-mediated signaling is crucial for Hydra regeneration.

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RESULTS Structural Analysis of the Whole Glycome of H. magnipapillata. To assess the structure of N- and O-glycans of H. magnipapillata, a mass spectrometry (MS)-based strategy was applied to released N- and O-glycans from complete tissue. The N-glycomic profile (Figure 1 and Figure 1 of the Supporting Information) was found to principally consist of 148

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fucosidases (α1−2, α1−3/4, and α1−6) were not successful, which is evident from MS profiles of fucosidase-treated samples (Figure 4 of the Supporting Information). However, the level of branching and the presence of antennae multifucosylation on H. magnipapillata N-glycans were corroborated by HF treatment. Underivatized fucosylated glycans, when treated with HF, result in rapid hydrolysis of Fucα1−3/4 linkages and slower release of α1−2-linked fucose, while the α1−6-linked fucose residues are relatively resistant.20 MALDI-TOF MS and MALDI-TOF/TOF MS/MS analysis of HF-treated released Nglycans demonstrated the following characteristics (Figure 5 of the Supporting Information): (i) a MS profile characterized by a complete loss of N-glycan structures above m/z 4800 as a result of the cleaved polyfucosylated residues and (ii) the presence of new peaks as a result of the cleaved fucose residues corresponding to bi-, tri-, tetra-, and penta-antennary structures with HexNAc−HexNAc branches on their antennae, mainly without any fucose (major in abundance) or to a lesser extent bearing residual fucose residues on their antennae or the proximal GlcNAc of the chitobiose core. Taken together, HF treatment verified the absence of poly-HexNAc−HexNAc antennae and the presence of polyfucose epitopes. We then conducted β-hexosaminidase digestion to determine the nature of the third (additional) HexNAc residue present on the less abundant N-glycan family [i.e., m/z 2990, 3164, 3512, 4074, and 4248 (Supporting Figure 1 of the Supporting Information)]. Underivatized, HF-treated N-glycans (Figure 5 of the Supporting Information) were enzymatically digested with a β-1−2,3,4-hexosaminidase and then subjected to MALDI-TOF MS analysis (Figure 6 of the Supporting Information). Unfortunately, the results showed that the additional HexNAc residue could not be digested, possibly because the residual fucose residues that appeared to be present on this antenna may be incompletely removed by HF treatment (Figure 7 of the Supporting Information). These results, however, verified that the GlcNAc residues beginning at the antennae, if not further substituted, were in a β-linkage [m/z 1416, 1906, and 2081 (compare Figures 5a and 6a of the Supporting Information)]. Permethylated N-glycans (neither subjected to HF treatment nor digested by fucosidase) were derivatized to partially methylated alditol acetates and subjected to linkage analysis by gas chromatography and mass spectrometry (GC−MS) (Table 1 of the Supporting Information). The detection of 2linked mannose was in accordance with the presence of biantennary N-glycans, while the detection of 2,4-linked, 2,6linked mannose and 2,4,6-linked mannose was in accordance with the presence of tri-, tetra-, and penta-antennary N-glycan structures. The presence of three predicted glycosyltransferase homologues, two GnT-IVs (XP_002161788.1 and XP_002159591.2) and one GnT-V (XP_002156726.2), also supported the detection of penta-antennary N-glycans. Terminal fucose, 2-linked fucose, and 4-linked fucose residues corroborated the presence of multifucosylated N-glycans. Results obtained from the linkage data described above combined with additional GC−MS linkage data obtained from the β-hexosaminidase-digested HF-treated N-glycans (Table 2 of the Supporting Information) suggested the presence of (i) LacdiNAc (GalNAcβ1−4GlcNAcβ1-) antennae (3-linked GalNAc, t-GalNAc, and 4-linked GlcNAc) and (ii) core fucosylation (4,6-linked GlcNAc). With regard to the O-glycans, MALDI-TOF MS and MALDI-TOF/TOF MS/MS analysis revealed that H. magni-

papillata consisted of core 1 and core 2 O-glycan structures bearing various amounts of fucose like the N-glycans (Figure 8 of the Supporting Information). GC−MS linkage analysis verified the presence of multiple fucose residues as well as the core 1 and core 2 sequences (Table 3 of the Supporting Information). Experimental Design for Studying the Functions of Novel Polyfucose Epitopes Containing Glycans during Regeneration. The various chemical, enzymatic, and mass spectrometry-based experiments described above established that the Hydra glycome is dominated by polyfucosylated, LacdiNAc antennae containing N- and O-glycans. To elucidate the biological role of these novel glycans in the regeneration of H. magnipapillata, we performed the following studies: (i) blocking or inhibiting the binding of polyfucosylated glycans to their cognate receptor(s) using fucose binding lectins during regeneration of amputated Hydra body parts and aggregated cell mass,17 (ii) rescuing regeneration with L-fucose monosaccharide, and (iii) altering the biosynthesis of fucosylated glycans by inhibiting fucosylation using a global metabolic inhibitor during regeneration of a Hydra (Scheme 1). Scheme 1. Experimental Design for Studying Glycan Functionsa

a

Regeneration of a Hydra is studied in two distinct methods: through classical regeneration of an amputed body part (a) and the other from aggregated cell mass (b). Both of these assays were used to study the glycan−receptor interactions in the presence of exogenous fuco-lectins and the global metabolic inhibitor of fucosylation.

Bioinformatics analysis of the Hydra genome using human ortholog FTs (α1−2, α1−3/4, and α1−6) led to the identification of 12 predicted FTs in Hydra. However, we did not find a gene corresponding to α1−2FucT in the genome of H. magnipapillata. The RNAi-based knockdown of FTs to elucidate the function of fucosylated glycans could not be undertaken because of the presence of a large number of putative FTs in Hydra and their unknown specificities. Incubation of H. magnipapillata Body Parts with Exogenous Fuco-lectins Inhibits Regeneration. Lectins have been used extensively to study glycan−receptor interactions. Here, we have used two α-linked L-fucose (LFuc) binding lectins, Ulex europeaus agglutinin-I (UEA-I, Hblood-specific) and Lotus tetragonolobus agglutinin (LTA, specific for LewisX and fucosylated LacdiNAc).21 However, 149

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Figure 2. Exogenous fuco-lectins block regeneration of body parts of H. magnipapillata. Hydra body parts (head, body column, and foot) were separately incubated at 18 °C with Hydra medium (control) or medium with lectins LCA, LTA, and UEA-I (10 μg/mL, 0.5 mL) in a 24-well plate. (a) Regeneration snap shots (48, 96, and 120 h) of control and LCA-, LTA-, and UEA-I-treated Hydra parts. Plot of the average (2 × 10) percentage of regenerated head from body columns (b) and foot-regenerated heads (c) incubated with Hydra medium alone (◆), LCA (■), LTA (▲), and UEA-I (●). Effect of LTA incubation time on Hydra regeneration. (d) Images of regenerating Hydra body parts on 7 days postamputation incubated with LTA (10 μg/mL, 0.5 mL of Hydra medium per well) for fixed time points (0, 24, 48, and 60 h) and then cultured in lectin-free medium. (e) Bar graph showing the average (2 × 10) percent regeneration of Hydra body columns (7 days postamputation) after they had been treated with LTA for 24, 48, and 60 h.

period. However, to address the necessity of continuous incubation, the regenerating body columns were incubated with LTA for only a defined period of time (control and 24, 48, and 60 h) and then changed to Hydra medium for the rest of the observation period. By 48 hpa, control Hydra body parts have developed hypostome and tentacle buds. However, there is a delay (of ∼24 h) in the regeneration of hypostome and tentacle buds from body columns and foot incubated with LTA for 24 and 48 h. Interestingly, 100% of the 60 h LTA-incubated body parts failed to regenerate either hypostome or tentacles or foot as shown in panels d and e of Figure 2. To probe further, the optimal concentration of LTA required to observe the lack-ofregeneration phenotype was studied by incubating Hydra body columns with either Hydra medium alone or varying concentrations of LTA (0.01, 0.1, 1.0, 5.0, and 10.0 μg/mL). The body columns soaked with 5 and 10 μg/mL LTA have regenerated (100%) neither head nor foot, whereas at lower LTA concentrations (0.01, 0.1, and 1 μg/mL), the regeneration was normal, albeit with formation of short and fewer tentacles at 0.1 and 1 μg/mL LTA in Hydra medium (Figure 11 of the Supporting Information). To support the hypothesis that the phenotype observed is due to the specific interaction of polyfucosylated glycans and endogenous receptor(s), we performed a rescue experiment with L-Fuc. First, the effect of L-Fuc alone under homeostatic and regeneration conditions was tested. All intact Hydra (2 × 5) treated with up to 100 mM L-Fuc in Hydra medium were normal under the condition of homeostasis. Approximately 60% of the body columns regenerated head and foot in the presence of 100 mM L-fucose. However, at lower L-Fuc concentrations (10, 25, and 50 mM), 100% of the body columns regenerated into whole Hydra (Figure 12 of the Supporting Information). Furthermore, none of the body columns regenerated either head or foot when the rescue experiments were conducted in the presence of LTA and L-Fuc (50 or 100 mM). Only above 50 mM L-Fuc was binding to endogenous fuco-lectin(s) possible, leading to modulation of signaling pathways that are important for regeneration. Thus, we hypothesized that the interaction between polyfucosylated

their specificity and affinity and avidity for polyfucosylated glycans are unknown. As a control, Hydra medium alone and a mannose binding lectin, Lens culinaris agglutinin (LCA), in Hydra medium were used. At first, binding of these lectins to Hydra was confirmed using LTA-FITC, UEA-FITC, and LCARhodamine. All three lectins bound equally to all the cell types (tentacles, hypostome, ectoderm, endoderm, and foot) as indicated by whole Hydra (live or fixed) staining (Figure 9 of the Supporting Information) and confirmed by cell macerates (data not shown). Next, the effect of these lectins on Hydra homeostasis was studied by incubating intact animals (five animals per well) with fluorescent lectins (10 μg/mL, as prescribed by suppliers) and Hydra medium (control) in a 24well plate. All animals treated with lectins looked normal even after incubation for 4 days at 18 °C (Figure 10 of the Supporting Information). For the regeneration experiments, fully grown Hydra were cut into three parts (head, body column, and foot from 10 animals) and incubated separately with Hydra medium (0.5 mL) or in the presence of fluorescently labeled lectins LCA, LTA, and UEA-I (10 μg/mL in Hydra medium) at 18 °C. Under the normal regeneration conditions, the hypostome appears at ∼48 h postamputation (hpa) and tentacle buds appear at ∼60 hpa followed by elongation of the tentacles and formation of peduncles (foot) by day 3. To our surprise, all of the 20 body columns (100%) incubated with LTA (10 μg/mL in Hydra medium) developed neither hypostome and tentacle buds nor foot even after day 3 and eventually disintegrated ∼120 hpa (Figure 2a−c). A similar lack of head and foot regeneration was also observed with foot and heads, respectively. However, UEA-I incubation showed only ∼30% of head (hypostome and tentacles) regeneration even after 3 dpa. The percentages of head and foot regeneration from foot and heads were ∼40 and ∼60%, respectively. While there was a delay in the percentage of regenerates incubated with LCA, there was no apparent defect in the regeneration of tentacles, hypostome, or foot. For the experiments described above, Hydra parts were incubated in the presence of lectins throughout the observation 150

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Figure 3. Fuco-lectins also inhibit reaggregation of Hydra from aggregates. Bright field images of control and LCA-, LTA-, and UEA-I-treated aggregated cell mass. While all the control and LCA-treated aggregates were able to regenerate into full grown Hydra by 96 h, 100% of LTA- and UEA-I-treated aggregates failed to regenerate.

glycans and endogenous fuco-lectin(s) might be inhibited in the presence of LTA and L-Fuc, leading to the complete shutdown of the signaling mechanism, which is important for regeneration of Hydra. Incubation with Fuco-lectins Also Blocks Regeneration of a Hydra from Aggregated Cell Mass. In addition to the classical regeneration experiments with Hydra body parts, as described earlier, the Hydra can also regenerate from dissociated cell mass.17 Dissociated, viable cells (∼2−5 × 105) were centrifuged to obtain a cell mass that was carefully transferred into a 24-well plate containing Hydra medium and observed for regeneration. To study the role of polyfucosylated glycans in the regeneration of a Hydra through the reaggregation assay, the cell pellets were incubated with lectins LTA and UEA-I (10 μg/mL in Hydra medium) and Hydra medium. In control experiments (five replicates), the cell aggregates became compact by 24 h, and the subsequent migration of endo- and ectodermal cells led to the formation of a bilayer cell structure as seen in adult Hydra. By 96 h, they form the body column, hypostome, and tentacle buds; however, cell masses incubated with both LTA and UEA-I failed to undergo compaction of cells, and eventually by ∼96 h, the cell mass started to disintegrate (Figure 3). Overall, these experiments confirm that the fuco-lectins were able to prevent regeneration of Hydra from either aggregated cell mass or

amputated body parts through inhibition of polyfucose− receptor binding. Effect of the Global Metabolic Inhibitor of Fucosylation during the Regeneration of Hydra. To support the hypothesis that inhibition or perturbation of polyfucosylated glycan−receptor interactions completely abolishes the regenerative capacity of the Hydra body columns, we used a chemical global metabolic inhibitor of fucosylation to remodel the Hydra glycan surface. Inhibitors for fucosyltransferases have been employed to elucidate the functions of fucosylated glycans both in vitro and in vivo.22,23 Recently, the effectiveness of 2Fperacetyl-fucose [compound 1 (Scheme 1)] as a global metabolic inhibitor of fucosylation has been demonstrated in CHO cells.24 Here, the same global metabolic inhibitor was used to remodel the Hydra glycome by inhibiting fucosylation in live animals. We first tested the toxicity of this global metabolic inhibitor 1 in Hydra cultures at different concentrations. Compound 1 was found to be safe at concentrations of up to 1 mM for at least a week. For the regeneration experiments, Hydra body columns (five animals) were incubated with control (0.1% DMSO in Hydra medium) and compound 1 (10, 200, and 500 μM and 1 mM) at 18 °C and observed for regeneration. By 4 days postamputation, the body columns under control conditions developed into fully grown animals with normal tentacles {average number of tentacles per 151

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Figure 4. Remodeling of glycans with the global metabolic inhibitor of fucosylation also inhibits Hydra regeneration. To inhibit the biosynthesis of polyfucosylated terminal epitopes on the glycans, the Hydra body columns were incubated with (a) Hydra medium with 0.1% DMSO and varying concentrations of 2F-peracetyl- fucose (1): (b) 10, (c) 200, (d) 500, and (e) 1000 μM. (f) Bar graph showing the average percentage of regenerated body columns (2 × 5 body columns, on day 5 hpa) as a function of compound 1 concentration. The error bars are standard deviations, obtained from two independent experiments using five animals each.

Hydra of 4.1 ± 0.3 [standard deviation (SD)] and length of 2.20 ± 0.73 mm (SD)}. On the other hand, only 4 out of 10 body columns treated with compound 1 (1 mM) regenerated hypostome and tentacles [average number of tentacles per Hydra of 1.3 ± 0.5 (SD) and length of 0.38 ± 0.09 mm (SD)]. Also, there was no peduncle development in these animals (Figure 4). Approximately 80% of body columns regenerated in 0.5 mM compound 1-treated body columns; however, as in 1 mM treated samples, the number of tentacles and their length were not normal [1.6 ± 0.8 (SD) and 0.27 ± 0.12 mm (SD), respectively]. Interestingly, buds developed from 0.5 mM compound 1-treated body columns have an average of four tentacles per bud. Furthermore, after day 5, the body columns incubated with both 0.5 and 1 mM compound 1 started to disintegrate, whereas control animals were normal. Approximately 95 and 100% of the body columns treated with 0.2 mM and 10 μM compound 1, respectively, have regenerated into whole Hydra without any apparent defects. A significant decrease in the level of binding of FITC-LTA with the global metabolic inhibitor for fucosylation-treated (0.5 and 1.0 mM) Hydra body columns suggests that compound 1 indeed inhibited the biosynthesis of polyfucosylated glycans (Figure 13 of the Supporting Information). These results corroborate our hypothesis that any perturbation in the polyfucosylated glycan−receptor interactions abolishes the regenerative capacity of the stem cell population. Identification of Putative Endogenous Fuco-lectin(s) via an Affinity Column and Nano-LC−MS/MS. To further support our hypothesis, we designed experiments to look for possible endogenous fuco-lectins in the Hydra proteome. For this study, fractions containing enriched glycopeptides of the trypsin digest of the whole Hydra proteome (from 250 Hydra) were obtained by following a protocol used for glycomics analysis (Figure 14 of the Supporting Information). The glycopeptides containing polyfucolysated N-glycans were further enriched using an LTA-bound agarose lectin column (Vector laboratories) and elution with buffer [50 mM HEPES (pH 7.4)] containing L-Fuc (3 mL of a 50, 100, or 200 mM solution). During this process, most high-mannose glycopeptides would have been removed. Fractions containing

predominantly polyfucosylated glycopeptides were concentrated and then chemically coupled with NHS-activated agarose dry resin beads under batch processing conditions [PBS (pH 7.4) for 2 h at room temperature]. The unreacted NHSactivated sites were quenched by passing 1 M Tris buffer (2 × 15 mL) through the polyfucosylated glycan-bound agarose column (gel volume of 7 mL). After the column had been coupled with glycopeptides, the binding property of the polyfucoslyated agarose column was ascertained using binding and elution of FITC-labeled LTA with buffer [2 × 7 mL of HEPES buffer (pH 7.4) with 10 μM Ca2+ and Mn2+] containing L-Fuc (10 and 100 mM). The presence of FITC-bound LTA was identified only in the 100 mM fraction using sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS− PAGE) (data not shown). To discover endogenous polyfucose-binding lectins, the whole Hydra proteome was obtained under nondenaturing conditions and the soluble and membrane fractions were separated using ultracentrifugation (Methods of the Supporting Information). Each of these fractions was separately loaded onto the polyfucosylated glycopeptide-bound agarose column, washed with 2 column volumes of buffer (50 mM HEPES with Ca2+ and Mn2+), and eluted with 2 column volumes (15 mL) of buffer containing L-Fuc (5, 100, and 150 mM) at 4 °C. Eluted fractions were separately concentrated to 500 μL using 3000 molecular weight cutoff Amicon filters, lyophilized, and then resuspended in water. Washes and eluates were analyzed using SDS−PAGE. Unique protein bands present in only 100 or 150 mM L-Fuc-containing eluates were cut (Figure 15 of the Supporting Information), and their identity was established using Nano-LC−MS/MS-based proteomics. Four unique proteins (Q6SIX6, Q2FBK2, Q2FBK4, and Q2FBJ9) were identified using five or more unique peptides. Q6SIX6 is a putative type II transmembrane C-type lectin, whereas the other three are putative peroxidases (PPODs). The SDS− PAGE band corresponding to PPODs is also present in the eluate containing 5 mM L-fucose. Hence, we believe that the PPODs do not bind strongly to L-fucose. Interestingly, PPODs have recently been shown to bind glycosaminoglycans.16 152

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DISCUSSION The glycome of Hydra presented here is characterized by the most heavily fucosylated N- and O-glycans reported to date. MALDI-TOF/TOF MS/MS, together with linkage analysis experiments, showed families of di-, tri-, tetra-, and pentaantennary structures whose antennae were comprised of GalNAc-GlcNAc sequences with varied degrees of fucosylation from one to four (Fuc4) on a single N-acetylhexosamine moiety (Figure 1 and Figure 2h of the Supporting Information). The βhexosaminidase digestion of HF-treated glycans (Figure 6a,b of the Supporting Information) has confirmed that each GlcNAc residue initiating an antenna is β-linked. Diagnostic elimination of mono-, di-, and tetrafucosides in CAD−MS/MS experiments (Figure 2a−h of the Supporting Information) suggests that the first fucose attached to both HexNAcs is 3-linked. Moreover, the major antennary structure of N-glycans of Hydra is GalNAcβ1−4GlcNAc (LacdiNAc). On the basis of the linkage analysis data of N- and O-glycans (Tables 1 and 3 of the Supporting Information), the proposed sequence of fucosides attached to the antenna is Fuc1−2Fuc and Fuc1−2Fuc1−2/ 4Fuc1−2/4Fuc. The MS and MS/MS data of the peak at m/z 7310 also confirmed that it corresponds to a tetra-antennary glycan with a remarkable total of 24 fucose units (Figure 2h of the Supporting Information). Interestingly, core fucosylation is absent in this molecule. In addition to these highly abundant complex structures, we have also observed a minor family of glycans whose antenna backbones are tri-HexNAc and are poorly fucosylated. The polyfucosylated glycans of Hydra closely resemble N-glycans of adult male Schistosoma mansoni.25 For example, the cercarial glycocalyx of S. mansoni consists of [(Fucα1−2)nGalNAc-(Fucα1−2)m-GlcNAc-Gal]n- epitopes in their O-glycans.26,27 These glycan epitopes are responsible for eliciting the immune reaction in humans.28,29 The binding of exogenous fuco-lectins (LTA and UEA-I) and LCA confirmed the functional availability of glycans as characterized by mass spectrometry. Significantly, a head-footless phenotype was observed during regeneration with body parts (Figure 2a), whereas binding of fuco-lectin to an intact Hydra did not have any effect. This is a unique type of phenotype that has not been reported so far in Hydra. More interestingly, under the conditions studied, incubation of LTA (≥5 μg/mL) at least 60 h was necessary to observe this phenotype (Figure 2d and Figure 11 of the Supporting Information). In addition to the classical regeneration assay, regeneration of a Hydra from cell mass also failed to occur in the presence of fucose binding lectins. Fuco-lectins LTA and UEA-I have not been reported to either activate or suppress proliferation. However, in vitro culturing of mouse blastocysts with LTA arrested the development through disruption of cell contacts, whereas UEA-I did not have any effect.30 A concentration-dependent, partial head and foot regeneration was also observed with a global metabolic inhibitor of fucosylation (Figure 4), suggesting the importance of fucose moieties and their interaction with receptors in Hydra regeneration. Although we cannot rule out an off-target effect of the global metabolic inhibitor of fucosylation, these results support our hypothesis that perturbation in the fucosylated glycan−receptor interactions probably abolished a downstream signaling cascade leading to regeneration defects of Hydra body columns. Our hypothesis is supported by the identification of putative endogenous fuco-lectins from the Hydra lysate through lectin

chromatography and mass spectrometry analysis. The type II transmembrane C-type lectin identified (protein id Q6SIX6_9CNID) belongs to sweet tooth proteins, with two carbohydrate recognition domains and a short N-terminal cytoplasmic tail. Recently, ∼22 putative sweet tooth family proteins in Hydra have been reported.16 Therefore, even though further experiments will be required to verify this hypothesis, we conclude from our pull-down experiments that an endogenous fuco-lectin is most likely the binding partner of the polyfucosylated glycans in Hydra. The data presented here support the hypothesis that polyfucosylated glycans and their cognate receptor-mediated inter- or intracellular signaling may be essential for head and foot regeneration of H. magnipapillata. A number of signaling pathways have been shown to be crucial for head and foot regeneration in Hydra. For example, Wnt signaling acts in axial patterning and head organization as shown by the early expression of genes such as HyTcf, HyβCat, and HyWnts (1−3, 7, 10, 11, and 16).31 HEADY, a short peptide, has also been shown to be important for head formation in Hydra.32 Furthermore, HEADY is not expressed in a head-less mutant reg-16 Hydra, confirming its role in head formation. Similarly, CnNK-2 and Shin Guard genes have been suggested in foot formation in Hydra. However, no single pathway has been implicated in controlling both head and foot formation. The data presented here show that disruption of polyfucose−receptor interactions affects both head and foot regeneration, suggesting its role in modulating several downstream processes in the regeneration of H. magnipapillata. The identification and establishment of molecular players downstream of fucolectin−receptor interactions are being pursued.

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METHODS

Hydra Culture. H. magnipapillata strain 105 was cultured in 1× Hydra medium [0.1 mM KCl, 1 mM CaCl2, 1 mM NaCl, 0.1 mM MgSO4 and 1 mM Tris−HCl (pH 8.0)]. This Hydra strain does not contain any known algal symbionts. All the experiments, including regeneration and reaggregation, were conducted at 18 °C, unless stated otherwise. The animals were starved for 2 days, and the growing buds were detached from their parents gently before the experiment. The Hydra were cut, to obtain the body column, head, and foot, such that the foot and head retain a part of the body column tissue, using a sterile scalpel. All images were recorded with an Olympus SZX16 microscope. Glycomics Analysis. Two hundred H. magnipapillata were freezedried and homogenized by sonication [with buffer containing 25 mM Tris, 150 mM NaCl, 5 mM EDTA, and 1% CHAPS (pH 7.4)], dialysis, reduction, carboxymethylation, and tryptic digestion as described previously.33 Peptide N-glycosidase F digestion of the tryptic glycopeptides was conducted in 50 mM ammonium bicarbonate (pH 8.5) for 20 h at 37 °C with 3 units of enzyme (Roche Applied Science). The released N-glycans were purified by using a Sep-Pak C18 cartridge (Waters Corp.) as described. The purified native N-glycans were either subsequently derivatized or subjected to modifications before derivatization. Before MALDI-TOF analyses, native N-glycans were derivatized using the sodium hydroxide permethylation procedure as described previously (Methods of the Supporting Information). Lectin Treatment of Amputed Parts of Hydra. For the regeneration experiments, body columns and head and foot regions of 10 animals were separately incubated in (500 μL) 1× Hydra medium alone or in Hydra medium containing lectins Rho-LCA, FITC-LTA, and FITC-UEA (10 μg/mL) in a 24-well dish, at 18 °C. They were observed for head and foot regeneration over a period of time. The medium with or without lectin was changed every third day. Body columns were observed for both head (hypostome and tentacle) and 153

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foot regeneration, whereas head and foot parts were observed for foot and head regeneration, respectively. Reaggregation and Regeneration of Dissociated Cells of Hydra. Hydra cells were dissociated according to the reported protocol with modifications.34 The head was removed from Hydra (200 numbers) were removed using a sterile scalpel and were washed thrice in 5 mL of dissociation medium 3.6 mM KCl, 6 mM CaCl2· 2H2O, 1.2 mM MgSO4·7H2O, 6 mM sodium citrate, 6 mM sodium pyruvate, 6 mM glucose, 12.5 mM HEPES (pH 7.1), 100 μg/mL streptomycin sulfate, and 50 μg/mL kanamycin (adjusted to pH 7.1)]}. After the final wash, the sample was left on ice for 30 min in 3 mL of dissociation medium. The Hydra were dissociated by gently pipetting up and down for 1 min and then allowed to sediment for 1 min, and this process was repeated three times. The supernatant was carefully transferred to a fresh 15 mL Falcon tube and centrifuged at 400g and 4 °C for 5 min in a swing rotor. The pellet was resuspended in 3 mL of dissociation medium, and viable cells were counted. Cell suspensions (2.5 × 105 cells/mL) were pelleted down in a 0.5 mL centrifuge tube at 400g and 4 °C for 10 min. Each pellet was carefully transferred into a 24-well plate containing dissociation medium alone (0.5 mL/well), LTA (10 μg/mL), or UEA-I (10 μg/mL). After 4 h, the culture medium was then diluted stepwise into Hydra medium, by adding 300 μL of Hydra medium to the respective wells (with antibiotics and lectins at the same concentration in dissociation medium). After 12 h, an additional 600 μL of Hydra medium was added to each well, and then media were replaced with fresh Hydra medium (with antibiotics and lectins at the same concentration in dissociation medium), 500 μL after 24 h. The medium was changed daily, and the aggregates were observed for regeneration. Rescue with L-Fucose. Known concentrations of L-fucose from Sigma-Aldrich (10, 25, 50, and 100 mM) were prepared in Hydra medium and incubated with Hydra body columns (duplicates of 10) at 18 °C and observed for regeneration. Time Course Exposure of the Amputated Hydra to FucoseSpecific LTA. The body column, head, and foot regions of Hydra were separately incubated in 1× Hydra medium only (control) or medium containing LTA lectin (10 μg/mL) for 12, 24, 48, and 60 h at 18 °C in a 24-well dish. After being incubated for 0 (control), 24, 48, and 60 h, the Hydra were washed and transferred into Hydra medium, and then their regeneration was observed. Regeneration with Different LTA Concentrations. The body columns of Hydra (duplicates of 5) were separately incubated in 1× Hydra medium only (control) or medium containing LTA (0.5 mL) at different concentrations (0.01, 0.1, 1.0, 5.0, and 10.0 μg/mL) at 18 °C in a 24-well dish. They were observed for regeneration and staining under the microscope. Effect of 2F-Peracetylfucose on the Regeneration of Hydra Body Columns. Hydra were starved for 2 days; duplicates of 5 each were incubated in 500 μL of 1× Hydra medium containing the global metabolic inhibitor at fucosylation concentrations of 10 μM, 200 μM, 500 μM, and 1 mM (with 0.1% DMSO). The medium was changed, and fresh medium containing inhibitors was added daily. The body columns were observed for regeneration. On day 6, the regenerating Hydra (global metabolic inhibitortreated and control, three from each set) were separately washed in Hydra medium and treated with 2% urethane until the Hydra were relaxed completely. They were then fixed using 4% paraformaldehyde (400 μL) for 1 h at room temperature, washed with Hydra medium, and then incubated in FITC-labeled LTA (10 μg/mL) containing Hydra medium (500 μL) for 2 h at 18 °C. The Hydra stained with lectins were then washed and observed under a microscope.

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biosynthetic pathway. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Y. Ghanekar, P. Sujana, and B. Hemalatha (inStem, Bangalore, India) for providing Hydra samples and discussions. We also thank Sai Sudha (inStem) for stimulating discussions and suggestions. We acknowledge H. Balaram (Jawaharlal Nehru Centre for Advanced Science and Research, Bangalore, India) for providing access to the MALDI-MS instrument. This work was supported by an inStem intramural grant (to S.R.) and grants from the Biotechnology and Biological Sciences Research Council (to A.D. and S.M.H.).

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ASSOCIATED CONTENT

S Supporting Information *

Additional information and supporting figures on the pull-down method, linkage analyses, MS/MS data analyses, α-fucosidase and HF treatment results, an O-glycan profile, images of fluorescently labeled lectin-bound Hydra, and the N-glycan 154

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